家蚕具有CHCH结构域的蛋白家族基因BmCH的原核表达和亚细胞定位  

Prokaryotic Expression and Subcelluar Localization of BmCH Gene with CHCH Structural Domain in the Silkworm,Bombyx mori

作　　者：舒建洪[1,2] 梁莉惠[1,2] 郑青亮[1,2] 牛伟涛[1,3] 张耀洲[1,2] 

机构地区：[1]浙江理工大学生命科学学院生物化学研究所,杭州310018 [2]浙江省家蚕生物反应器和生物医药重点实验室,杭州310018 [3]邢台学院生化系,河北邢台054001

出　　处：《蚕业科学》2011年第2期238-245,共8页ACTA SERICOLOGICA SINICA

基　　金：浙江省自然科学基金项目(No.Y3080183;Y3090176);浙江省"生物医学工程"重中之重学科开放基金项目(No.SWYX0909;SWYX0910)

摘　　要：含有CHCH保守结构域的蛋白在不同物种中行使不同的功能。从家蚕蛹cDNA文库筛选获得一条cDNA序列,经生物信息学分析发现其编码蛋白含有一个CHCH保守结构域,将该基因命名为BmCH(GenBank登录号:DQ443425.1)。该序列开放阅读框大小为435 bp,编码144个氨基酸残基。将成功构建的重组质粒pET-28a(+)-BmCH转化到E.coli Rosetta(DE3)菌株,经IPTG诱导表达BmCH融合蛋白,用亲和层析进行纯化后,将融合蛋白免疫新西兰大白兔制备多克隆抗体。通过Westernblotting检测BmCH在家蚕4个发育时期和5龄幼虫组织中的表达情况与用荧光定量PCR检测BmCH在家蚕不同发育时期及5龄幼虫组织中的转录结果基本一致:该蛋白在卵、幼虫、蛹、蛾4个发育时期均有表达,其中在5龄幼虫期的表达量最高;该蛋白在5龄幼虫的表皮、精巢、中肠、丝腺和卵巢中有明显表达,在血淋巴、马氏管、脂肪体中的表达量较少,但差异不大,而在气管和头部没有表达。利用抗体的免疫荧光标记对BmCH在家蚕BmN细胞中的定位进行研究,观察到该蛋白主要分布在细胞质中。初步推测BmCH可能是家蚕生长发育的必需蛋白,参与多种细胞生命活动的过程。Proteins containing CHCH conserved domain play different roles in various organisms.A cDNA sequence was obtained from screening the Bombyx mori pupa cDNA library constructed in our laboratory.Sequence analysis showed that it encodes a protein containing the CHCH domain.We preliminary nominated this novel gene as BmCH（Bombyx mori CH）（GenBank accession number DQ443425.1）.It contains an open reading frame of 435 bp and encodes 144 amino acids.The BmCH gene was cloned into the prokaryotic expression vector pET-28a,and the recombinant plasmid pET-28a-BmCH was transformed into E.coli Rosetta（DE3）.After IPTG induction,the BmCH fusion protein was expressed and purified with metal-chelating affinity chro-matography.The purified fusion protein was used to immunize New Zealand rabbit for preparing polyclonal antibodies.BmCH protein expression detected by Western blotting and transcriptional level of BmCH gene detected by real-time fluorescent quantitative RT-PCR in Bombyx mori larvae of 4 developmental stages and in various tissues of the 5th instar larvae were consistent.It was found that BmCH protein was expressed in all four stages,i.e.egg,larva,pupa and moth stages,among which the highest level was in larva of the 5th instar.Obvious expression of this protein was detected in epidermis,testis,midgut,silk gland,and ovary.Low level expression was observed in hemolymph,Malpighian tubule,and fat body.No expression was observed in trachea and head.Subcellular localization with immuno-fluorescent antibody against BmCH protein showed that this protein was mainly localized in the cytoplasm,suggesting that BmCH protein may be an essential protein to the growth and development and participates in life activities of different cells in B.mori.

关 键 词：家蚕 CHCH保守结构域 基因克隆 荧光定量PCR Westernblotting分析 细胞定位 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]